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1. Field of the Invention
The present invention relates to a composition for preserving determined and reproducible amounts of micro-organisms.
It relates further to lens-shaped pellets comprising such a composition.
It has also as an object a process for producing such pellets.
2. Description of Related Art
The presence of benzene in mineral water Perrier, Listeria in cheese or epizooty of bovine spongiform encephalopathy have made quite a stir. The companies in the field of water, foodstuffs or health, positioned in sensitive sectors, have customers quite attentive to the quality of the goods delivered to them. Thus, the prevention of crisis situations is vital. In fact, any incident, such as a contamination, may have serious consequences for companies (waste production, image degradation). Consequently the danger of a contamination is a major concern for the authorities who edict increasingly stronger national, European or international regulatory constraints in terms of microbiological safety and for the industries who establish quality assurance plans including in particular the search of critical contamination points in the processes.
Consequently quite a number of public or private laboratories carry out each year many thousands of chemical and microbiological analysis in the fields of foodstuffs, environment or health.
Inter-laboratory studies have clearly showed that the laboratories generally worked with quite a good precision, but important variations existed between the results obtained from different laboratories.
Thus, excess results may be observed in laboratories having badly adjusted stoves, whereas default results are observed in laboratories having culture media of a poor quality.
Such erroneous results may have an great economical impact upon public health. The excess results may lead to refuse wrongly a product batch, to programme unnecessarily sanitizing operations to improve a beach quality or to disturb people by prohibiting tap water consumption.
In contrast, default results may incur a risk for the consumers by authorizing sales of contaminated products, distribution of poorly disinfected water or bathe in polluted water.
To secure reliable measurements, the checking laboratories have been asked to be accredited. Initially such accreditation only defined obligations of means (qualified personnel, adapted rooms, maintenance of measurement apparatus, control of raw materials and writing of the analytical procedures). At the present time, it also requires obligations of results. Such a new requirement imposes the implementation of a quality system including internal controls and participation to inter-laboratory tests.
For controlling the analytical chains internally and organize inter-laboratory tests, it is imperative to use reference materials. Such reference materials must be stable in time and should allow to reconstitute homogenous control samples containing an exact amount of the product to be dosed.
In chemistry, such reference materials have been existing for many years. In contrast, in the microbiological field, it is particularly difficult to preserve exact amounts of micro-organisms. In fact, the number of micro-organisms may either increase, if the preservation media enables their multiplication during manufacture, transport or storage, or decrease, if they die during manufacture, transportation or storage or at the time of the reconstitution of the control sample before use.
Methods for preserving micro-organisms, such as lyophilization and cryoconservation, have been already described in the all. However, such methods lead upon the conditioning step to high mortality rates amongst the micro-organisms being preserved, and consequently do not allow to master accurately the final quality of the surviving micro-organisms.
Thus, two patents appealing to a rude freezing method in liquid nitrogen do not describe the preservation of bacteria or micro-organisms and do not address to the problem of the quantitative dosages.
U.S. Pat. No. 5,364,756 discloses a method of cryoconservation for eucalyote cells. A suspension of such cells is prepared in a buffer comprising cryoprotecting agents, and then the solution is nebulizated with ultrasounds so as to form microdroplets that are rapidly cooled and dried.
U.S. Pat. No. 5,405,616 relates to the preservation of pharmacologically active molecules. The particles comprising such molecules are predominantly made of a skeleton-forming water-soluble hydrophilic macromolecule. Various proteins are mentioned, mainly proteins from vegetal origin.
Exact amounts of micro-organisms may be obtained from naturally or artificially contaminated samples. However, the preservation of such samples cannot go beyond 24 hours and requires a lower temperature than 10xc2x0 C.
Exact amounts of various bacteria are also obtained after freezing in a medium containing serum and inositol (1993 Peterz and Steneryd, J. of Appl. Bacteriol., 74, 143-148) or skimmed milk (1994 Schijven, Havellaar and Bahar, Appl. And Environ. Microbiol., 60, 4160-4162). It seems however that such frozen bacterial suspensions are not stable beyond three months for the first type of preparation and one year for the second one. Moreover, they require very demanding transportation conditions (short deliveries and maintenance of the temperature below xe2x88x9270xc2x0 C.). Finally their stability in case of successive thaw-freeze has not been demonstrated.
Presently only atomization manages to conciliate the preservation, stability and transportation constraints with the requirement of an exact amount of micro-organisms.
Suspensions of Bacillus cereus in concentrated milk atomized and encapsulated in gelatine have been used as reference materials after transportation at room temperature (Paul H. In""t Veld, 1993, Int. J. of Food Microbiol., 20, 23-36).
However, such technique is difficult to be implemented, because it requires a dissolution of the gelatine in a reconstitution medium maintained a 37xc2x0 C. Moreover, some species like Aeromonas hydrophila, Pseudomonas aeruginosa and Campylobacter jejuni cannot be preserved stably by atomization. Such a process is not recommended either for preserving respiratory tract pathogens being transmissible with aerosols like Legionella pneumophila. Finally the reference materials prepared by atomization have a high cost.
It appears thus from the art that no process was known which would be easy to be carried out and cheap allowing to preserve stably exact amounts of micro-organisms.
The Assignee has solved this problem by finding a particular composition being able to hold viability of micro-organisms.
The present invention thus relates to a composition for preserving predetermined and reproducible amounts of micro-organisms comprising in combination efficient amounts of at least one material being able to form the skeleton of lens-shaped pellets and at least one saturating material as well as micro-organisms.
For the present invention it is meant by reproducible amounts of micro-organisms variations of the ratio between two measurements comprised, in 95% of the cases, between 0.25 and 4, more preferably between 0.5 and 2.
Said composition comprises from about 10 to 60% of substances being able to form the skeleton of the lens-shaped pellets. Preferably a mixture of albumin and starch is used containing from about 20 to 40% albumin and from 2 to 5% starch (in total weight of the composition). After freezing and drying, these molecules form a highly porous and simultaneously mechanically stable skeleton that is dissolved rapidly in water.
Advantageously albumin belonging to such a composition is ovalbumin. Ovalbumin may appear in particular as an egg white preparation. Egg white is a sterile medium rich in protecting proteins within which it is possible to disperse the micro-organisms homogeneously. It may be however any other albumin (bovine albumin serum) or any protein having the same function.
Starch can be any type of starch and, in particular, natural as well as modified starches, dextrans, dextrins and maltodextrin. It can be substituted by any other hydrophilic macromolecule, particularly vegetal proteins or the hydrolysates thereof, collagens, gelatins, elastin hydrolysate or mixtures of above-mentioned substances.
Said composition also comprises from about 40 to 90% of a saturating substance. The saturating substance may be formed with one or more mixed salts or sugars. Such salts or sugars may be added as powder, brine or syrup. By using saccharose, preferably from about 60 to 80%, the composition has a lower water activity than 0.9. Consequently, the cellular damages occasioned by freezing and drying are limited and the development of micro-organisms is prevented in particular upon any transportation at room temperature.
According to one embodiment of the invention, the composition contains one or more types of micro-organisms. They can be strict anaerobic bacteria, aero-anaerobic or strict aerobic bacteria, psychrophilic bacteria, mesophilic or thermophilic bacteria, halophilic bacteria, enterobacteria, streptococci, staphylococci, pathogenic bacteria (Salmonella, Campylobacter, Yersinia, Listeria, Pseudomonas aeruginosa, Aeromonas, Vibrio, etc.), yeasts or moulds or fungi, bacteriophages or viruses, or protozoal cysts.
Advantageously, the present composition includes from 102 to 1011, preferably from 102 to 109 micro-organisms per gram. Such a composition may be advantageously presented as pellets and in particular as lens-shaped pellets having a diameter in a range of 1 to 10 mm.
Such pellets have advantageously a mass from about 1 to 250 mg, preferably from about 2 to 100 mg, more preferably from 10 to 25 mg.
The pellets according to the present invention can be advantageously obtained with a manufacturing process comprising the following steps of:
a) incorporating micro-organisms into the skeleton material,
b) reducing water activity by adding progressively the saturating substance,
c) shaping the pellets, and
d) drying the pellets under vacuum and at a temperature lower than about xe2x88x9210xc2x0 C.
Preferably the mixture is carried out as follows:
a) mixing albumin and micro-organisms,
b) adding starch, and then
c) adding saccharose.
Various drying modes are to be envisaged. Advantageously pellet drying is carried out in a desiccator in a period from about 12 hours to 10 days.
In view of an optimal stability, i.e. over a period of more than 12 months, the pellets may be stored at xe2x88x9270xc2x0 C. in the presence of a dehydrating bag. They can however be preserved 3 months at xe2x88x9220xc2x0 C. and 4 days at room temperature, making thus for example their transport easier since no particular and complexed transport condition is required.
A reconstitution medium convenient for a use in the present invention should limit at a maximum the osmotic shock upon the pellet dissolution. A 23 g/l synthetic sea salt solution is recommended as a reconstitution medium, since it presents a higher recovering level of the micro-organisms than the diluents usually used in microbiology such as Ringer, saline peptone or distilled water. Such a solution may be the one sold by Aquarium Systems (MENTOR, Ohio, USA) under the name Instant Ocean, or the DSM medium sold by SANOFI PASTEUR (Marnes la Coquette, France).
Pellet dissolution should be effected at room temperature. If the sample is not analyzed within half an hour after the reconstitution, it is to be recommended to maintain it in melting ice so as to guarantee a good stability.
Such pellets containing micro-organisms stable over time, homogeneous and able to be reconstituted as a suspension reproducibly, i.e. with amounts varying weakly from one test to another one, constitute actual reference materials particularly adapted for checking the reliability of measurements (internal and external quality control) in the bacteriology field for waters, drinks and foodstuffs generally, in pharmacy, cosmetics and environment.
This type of reference material may also be used advantageously for xe2x80x9cchallenge testsxe2x80x9d in the foodstuffs field. For such a specific use, the pellets contain one or more micro-organisms. Pellets may be directly introduced into the product to be tested (alu-dish, salad bag, cooked dish, etc.). The products are then put in real preservation conditions and the progress of the contamination introduced quantitatively into the products is monitored according to the usual numbering procedures.
More generally, this new type of reference material may be used for the quantitative microbiological analysis:
checking the presence/absence of micro-organisms,
checking the fertility of culture media,
checking the efficiency of antibiotics, bactericide or bacteriostatic substances,
checking sterilization (ultraviolet, chloration, ozonization, filtration, etc.).
According to another aspect of the invention, the pellets containing at least one type of micro-organisms may be advantageously used in specific products as starting agents for seeding the species so as to master a crucial microbiological parameter interfering in the fermentation process.
Thus, pellets containing yeasts, such as for example S. cerevisae, S. calbergensis, S. ellipsoidus, may be used to produce bread, high and low pressure beers, wine and other alcohols.
Pellets containing lactic bacteria may advantageously be used to produce fermented milks and yoghurt as well as in the pharmaceutical industry to produce products convenient for re-seeding intestinal flora.
Pellets containing activated fungal spores may be used directly or after dilution to a desired content of micro-organisms, in the foodstuffs field, particularly for cheese or salted food products. Said compositions may for example be spread or incorporated into the products to be converted, such as cheeze of salted food products.
Thus, pellets containing specific bacterial species, such as for example Rhizobium, Pseudomonas, Bacillus, Arthrobacter, Serratia and Azospirillum, may be used in agriculture and in the environment industry.
The present invention will be now illustrated without being limited by the following examples.